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Molecular Endocrinology 18(10):2451–2462 Copyright © 2004 by The Endocrine Society doi: 10.1210/me.2004-0173
Small Ubiquitin-Like Modifier 1 (SUMO-1) Modification of the Synergy Control Motif of Ad4 Binding Protein/Steroidogenic Factor 1 (Ad4BP/SF-1) Regulates Synergistic Transcription between Ad4BP/SF-1 and Sox9 TOMOKO KOMATSU, HIROFUMI MIZUSAKI, TOKUO MUKAI, HIDESATO OGAWA, DAICHI BABA, MASAHIRO SHIRAKAWA, SHIGETSUGU HATAKEYAMA, KEIICHI I. NAKAYAMA, HIDEKI YAMAMOTO, AKIRA KIKUCHI, AND KEN-ICHIROU MOROHASHI Department of Developmental Biology (T.K., H.M., T.M., H.O., K.-I.M.), National Institute for Basic Biology, Okazaki 444-8787, Japan; School of Life Science (T.K., K.-I.M.), The Graduate University for Advanced Studies, Okazaki 444-8585, Japan; Core Research for Evolutional Science and Technology (CREST) (H.M., S.H., K.I.N., K.-I.M.), Japan Science and Technology Corporation, Kawaguchi 332-0012, Japan; Graduate School of Integrated Science (D.B., M.S.), Yokohama City University, Yokohama 2300045, Japan; Department of Molecular and Cellular Biology (S.H., K.I.N.), Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan; and Department of Biochemistry (H.Y., A.K.), Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan An orphan nuclear receptor, Ad4 binding protein/ steroidogenic factor 1 (Ad4BP/SF-1), is essential for the development and function of steroidogenic tissues. To examine the transcriptional regulation of Ad4BP/SF-1, two-hybrid screening was performed, and the sumoylation [conjugation of a small ubiqutin-like modifier (SUMO-1)] components Ubc9, protein inhibitor of activated STAT 1 (PIAS1), and protein inhibitor of activated STAT 3 (PIAS3) were isolated. Cultured cell and in vitro studies revealed that Ad4BP/SF-1 is sumoylated at K119 and K194. Because K194 lies within the synergy control (SC) motif defined to repress synergistic transcription from promoters containing multiple binding sites, correlation between the functions of the SC motif and sumoylation was investigated. The K194R mutant of Ad4BP/SF-1, which cannot be sumoylated, showed enhanced synergistic transcription from a promoter containing multiple Ad4/SF-1 sites, suggesting that sumoylation is necessary for repression of transcriptional synergy through the SC motif. It has been estab-
lished that the Mu¨llerian inhibiting substance gene is transcribed predominantly under the control of Ad4BP/ SF-1 and, moreover, its transcription is regulated synergistically with Sox9, Gata4, and Wt1. Interestingly, it was found that all of these factors are sumoylated, and these sumoylation sites occur within SC motifs. Based on the observation that SC motif mutants of Ad4BP/ SF-1 and Sox9 resulted in the enhancement of their synergistic transcription, it was concluded that the SC motif regulates synergistic transcription even between distinct types of transcription factors. Considering that both mutants cannot be sumoylated, it is likely that sumoylation is implicated in this regulation. Because it was revealed with an in vitro sumoylated Ad4BP/SF-1 that DNA binding activity and interaction with Sox9 were unaffected, sumoylation may regulate transcription through affecting selective and cooperative interaction among factors constituting transcriptional complexes. (Molecular Endocrinology 18: 2451–2462, 2004)
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transcription factor regulating steroidogenic genes (1, 2). Subsequent gene disruption studies clearly showed that Ad4BP/SF-1 plays crucial roles in the development of endocrine tissues such as the adrenal gland, gonads, and pituitary as well as nonendocrine tissues such as the ventromedial hypothalamus and spleen (3–6). Because of its functional significance, molecular mechanisms underlying the transcriptional regulation of Ad4BP/SF-1 have been investigated by focusing on its protein interactions and posttranslational modifications. Indeed, a number of coregulators such as steroid receptor coactivator 1 (SRC-1) (7, 8), cAMP response element-binding protein (CREB)binding protein/p300 (9), transcriptional intermediary factor 2 (TIF2) (10), nuclear receptor corepressor (N-
D4 BINDING PROTEIN/steroidogenic factor 1 (Ad4BP/SF-1, NR5A1), a member of a nuclear receptor superfamily, was originally identified as a Abbreviations: Ad4BP/SF-1, Ad4 binding protein/steroidogenic factor 1; DBD, DNA binding domain; DTT, dithiothreitol; GFP, green fluorescent protein; GST, glutathione-S-transferase; HEK, human embryonic kidney; HDAC, histone deacetylase; IPTG, isopropyl--D-thiogalactopyranoside; LBD, ligand binding domain; Mis, Mu¨llerian inhibiting substance; PIAS, protein inhibitor of activated STAT (signal transducer and activator of transcription); SC, synergy control; SCF, synergy control factor; SUMO, small ubiquitin-like modifier; tk, thymidine kinase. Molecular Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community.
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CoR) (11), and -catenin (12) have been reported to interact with Ad4BP/SF-1, whereas multiple types of transcription factors such as Sox9 (13), Gata4 (14), Wt1 (15, 16), PITX1 (17), multiprotein bridging factor 1 (MBF1) (18), EGR1 (19, 20), and TReP-132 (21) have been reported to show transcriptional synergy with Ad4BP/SF-1 through direct interaction. In addition, dosage-sensitive sex reversal-adrenal hypoplasia congenital critical region on the X chromosome, gene 1 (Dax-1) (22), DP103 (23), androgen receptor (24, 25), and zinc finger protein 67 kDa (Zip67) (26) have been shown to repress the transcriptional activity of Ad4BP/SF-1. With respect to posttranslational modification, it was shown that phosphorylation of Ser 203 by MAPK activates the transcription of Ad4BP/SF-1 (27, 28), whereas protein phosphatase is also involved in transcriptional regulation (29). Finally, it was reported that GCN5 modulates the transcriptional activity of Ad4BP/SF-1 through acetylation (30). Recently, a novel posttranslational modification, sumoylation [conjugation with a small ubiqutin-like modifier (SUMO)], has been identified to play a crucial role in a variety of cellular processes (31–33). SUMOs (SUMO-1, SUMO-2, and SUMO-3) are structurally related to ubiquitin and are ligated to lysine residues by stepwise enzymatic reactions similar to ubiquitination. It has been established that SUMOs are synthesized as precursor forms and undergo maturation through proteolytic cleavage to expose a carboxy-terminal glycine residue. The mature forms are activated by an E1 enzyme, Aos1/Uba2, and successively transferred to the E2 conjugation enzyme, Ubc9. Finally, target proteins are sumoylated through the action of E3 enzymes such as PIAS (protein inhibitor of activated STAT) family members, RanBP2, and PC2. Although it has been suggested that sumoylation affects protein-protein interaction (34, 35), alters subcellular and intranuclear localization of its target proteins (36), and antagonizes other modifications (37, 38), the functions of sumoylation remain controversial. Likewise, no consensus exists regarding the effects of sumoylation on transcriptional activity, because sumoylation has been reported to enhance or otherwise repress transcription (39, 40). Recently, however, the consensus sequence for sumoylation was found to be included in the synergy control (SC) motif, which is conserved in the suppression domains of a variety of transcription factors and is essential for the regulation of synergistic transcription (41). Moreover, it was indicated that the function of the SC motif is regulated by sumoylation (42, 43). In this study, we demonstrate that Ad4BP/SF-1 is sumoylated at two lysine residues and that one of these sumoylation sites lies within an SC motif. Mutation of the SC motif leads to an enhancement of synergistic transcription from a promoter containing multiple Ad4/SF-1 sites. In addition, SC motif regulates synergistic transcription of the Mis (Mu¨ llerian inhibiting substance) gene between Ad4BP/SF-1 and Sox9, the latter of which is also sumoylated within its SC motif. These findings suggested that sumoylation of Ad4BP/
Komatsu et al. • Regulation of Ad4BP/SF-1 by SUMO
SF-1 within the SC motif is involved in regulation of transcriptional synergy.
RESULTS Identification of PIAS1 and PIAS3 as Proteins Interacting with Ad4BP/SF-1 To study the regulation of the function of Ad4BP/SF-1, we performed a yeast two-hybrid screen using a library prepared from mouse fetal gonads. When fulllength Ad4BP/SF-1 was used as a bait plasmid, a number of positive clones, including Ubc9, PIAS1, and PIAS3, were isolated. The isolated PIAS3 corresponds to the long form recently reported as PIAS3L (44). The interaction between Ad4BP/SF-1 and PIAS1 or PIAS3 was further confirmed by coimmunoprecipitation assays with transfected human embryonic kidney (HEK) 293 cells. HEK 293 cell lysates containing green fluorescent protein (GFP)-Ad4BP/SF-1 and 3xFlag-PIAS1 or 3xFlag-PIAS3 were subjected to immunoprecipitation with anti-Flag antibody, and the precipitates were analyzed by Western blotting with an anti-Ad4BP/SF-1 antibody. As shown in Fig. 1A, GFP-Ad4BP/SF-1 coimmunoprecipitated with an anti-Flag antibody only in the presence of 3xFlag-PIAS1 and 3xFlag-PIAS3. Reciprocally, 3xFlag-PIAS1 and 3xFlag-PIAS3 were coimmunoprecipitated with GFP-Ad4BP/SF-1 (Fig. 1B). The region in Ad4BP/SF-1 responsible for the interaction with PIAS3 was investigated by coimmunoprecipitation experiments using full-length and truncated forms of GFP-Ad4BP/SF-1 as shown in panel (a) of Fig. 1C. The immunoprecipitates with anti-GFP antibody were analyzed by Western blotting to detect 3xFlag-PIAS3. 3xFlag-PIAS3 coimmunoprecipitated efficiently with full-length, 1–129, and 1–78 constructs of GFP-Ad4BP/SF-1. The other truncated forms lacking the N-terminal 78 amino acids failed to interact with PIAS3, indicating that the N-terminal region corresponding to the Zn finger DNA binding domain (DBD) of Ad4BP/SF-1 is sufficient for its interaction with PIAS3. Likewise, Ad4BP/SF-1 interacted with PIAS1 through the same N-terminal 78 amino acids (data not shown). Sumoylation of Ad4BP/SF-1 in Vivo and in Vitro Because PIAS family members have been reported to function as SUMO E3 ligases, we tested the possibility that Ad4BP/SF-1 is a target for sumoylation. Ad4BP/ SF-1 was transfected into HEK 293 cells in the presence or absence of GFP-SUMO-1 or GFP-SUMO-2, and the cell lysates were subjected to Western blotting with anti-Ad4BP/SF-1 antibody (Fig. 2A). When Ad4BP/SF-1 was expressed alone, two slowermigrating bands at 70 and 73 kDa (indicated by an open arrowhead) were detected in addition to the 53kDa unmodified Ad4BP/SF-1 (indicated by an arrow). As described below, the signal at 73 kDa was con-
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firmed as a sumoylated Ad4BP/SF-1, whereas the 70kDa band remains of obscure origin. Moreover, a weak band was often observed at 90 kDa (Fig. 3B, indicated by double open arrowheads). In the presence of GFPSUMO-1 or GFP-SUMO-2, two bands were additionally observed at approximately 150 kDa and 250 kDa (indicated by single and double closed arrowheads, respectively). Coexpression of PIAS3 and PIAS1 increased the intensity of these two bands (Fig. 2B). Thus, these two bands were interpreted to be sumoylated forms of Ad4BP/SF-1. To confirm this, the HEK 293 cell lysates transfected with 3xFlag-Ad4BP/SF-1 in the presence or absence of GFP-SUMO-2 were subjected to immunoprecipitation with an anti-Flag antibody, and the immunoprecipitates were analyzed by Western blotting with anti-Ad4BP/SF-1, anti-GFP,
Fig. 1. Interaction of Ad4BP/SF-1 with PIAS through DBD A, Pull-down assay of Ad4BP/SF-1 by PIAS. Expression plasmids for GFP-tagged Ad4BP/SF-1 and 3xFlag-tagged PIAS1 or PIAS3 were transfected into HEK 293 cells. Total cell lysates were subjected to immunoprecipitation with antiFlag M2 antibody-agarose (␣-Flag). Coimmunoprecipitated GFP-Ad4BP/SF-1 was detected by Western blotting with anti-Ad4BP/SF-1 antibody (␣-Ad4BP). As a control, Ad4BP/ SF-1 in 2% of the total cell lysates is indicated. B, Pull-down assay of PIAS by Ad4BP/SF-1. Transient transfection was performed with the expression plasmids as indicated in panel A. Total cell lysates were subjected to immunoprecipitation with anti-GFP antibody (␣-GFP) and coimmunoprecipitated 3xFlag-PIAS1, or PIAS3, was detected with anti-Flag antibody. C, Determination of a region in Ad4BP/SF-1 responsible for interaction with PIAS. Wild-type and truncated forms of GFP-Ad4BP/SF-1 used in this assay are indicated schematically in (a). The numbers indicate the amino acid residues for Ad4BP/SF-1. The region essential for interaction with PIAS is indicated by a horizontal line at the bottom. These truncated forms of GFP-Ad4BP/SF-1 were transfected with 3xFlag-PIAS3 into HEK 293 cells, and the cell lysates were subjected to immunoprecipitation with anti-GFP antibody (b). Coimmunorecipitated 3xFlag-PIAS3 detected with anti-Flag antibody (top panel) and 3xFlag-PIAS3 in 2% of the total cell lysates (middle panel) are shown. Recovery of the truncated forms of Ad4BP/SF-1 was confirmed with anti-GFP antibody (bottom panel).
Fig. 2. Detection of Sumoylated Forms of Ad4BP/SF-1 in Cultured Cells A, Detection of sumoylated forms of Ad4BP/SF-1. Expression plasmids for Ad4BP/SF-1 and GFP-SUMO-1 or GFPSUMO-2 were transiently transfected into HEK 293 cells. Total cell lysates were immunoblotted with anti-Ad4BP/SF-1 antibody. B, Enhanced sumoylation by PIAS1 and PIAS3. Expression plasmids for Ad4BP/SF-1 and GFP-SUMO-1 were transfected into HEK 293 cells in the presence or absence of 3xFlag-PIAS3 and 3xFlag-PIAS1. Ad4BP/SF-1 in total cell lysates was detected by anti-Ad4BP/SF-1 antibody (top panel). Expression of 3xFlag-PIAS1 and 3xFlag-PIAS3 was confirmed with anti-Flag antibody (bottom panel). C, Confirmation of sumoylated products with antibodies to Ad4BP/SF-1, GFP, and SUMO. Expression plasmid for 3xFlag-tagged Ad4BP/SF-1 was transfected into HEK 293 cells in the presence or absence of GFP-SUMO-2. After total cell lysates were immunoprecipitated with anti-Flag antibody, the precipitates were examined with anti-Ad4BP/SF-1 (left; with ␣-Ad4BP), anti-GFP (middle; with ␣-GFP), and antiSUMO-2/3 antibodies (right; with ␣-SUMO-2/3). Arrow and open arrowhead indicate Ad4BP/SF-1 unmodified and modified with endogenous SUMO, respectively. Single and double closed arrowheads indicate Ad4BP/SF-1 associated with GFP-SUMO at one and two lysine residues, respectively. Locations of molecular weight markers are indicated on the left of the panels.
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Fig. 3. Ordered Sumoylation of Ad4BP/SF-1 from Lysine 194 to Lysine 119 A, Schematic representation of Ad4BP/SF-1. Numbers indicate amino acid residues. DBD, putative LBD, and the location of the two sumoylation sites are shown. B, Disappearance of sumoylation signal from KR mutants of Ad4BP/ SF-1. Expression plasmids for KR mutants, K119R, K194R, and 2KR, were transfected into HEK 293 cells in the absence (left) or presence (right) of GFP-SUMO-1. Ad4BP/SF-1 in total cell lysates was detected by anti-Ad4BP/SF-1 antibody. Single and double open arrowheads indicate sumoylated Ad4BP/SF-1 with endogenous SUMO (left) whereas single and double closed arrowheads indicate sumoylated Ad4BP/ SF-1 with transfected GFP-SUMO-1 (right).
and anti-SUMO2/3 antibodies. As shown in Fig. 2C, in the absence of GFP-SUMO-2, the anti-Ad4BP/SF-1 antibody detected a band at 73 kDa, probably representing a protein modified with an endogenous SUMO molecule, whereas in the presence of GFP-SUMO-2, the antibody detected an additional sumoylated signal at 150 kDa. This 150-kDa signal was also detected with anti-GFP and anti-SUMO-2/3 antibodies. These results indicate that Ad4BP/SF-1 is a sumoylation target, and that PIAS1 and PIAS3 can promote sumoylation of Ad4BP/SF-1 in vivo. Subsequently, we searched Ad4BP/SF-1 for the consensus sequence for sumoylation, ⌿KXE, where ⌿ is a hydrophobic and X is an unspecified amino acid residue, and found that two lysine residues, K119 and K194, occur within consensus sequences (Fig. 3A). Thus, we tried to determine whether mutant forms of Ad4BP/SF-1 containing an arginine substitution at K119, K194, or both lysine residues can be sumoylated. Western blot analyses revealed that the 90-kDa band observed with the wild-type Ad4BP/SF-1 disappeared in the K119R mutant, whereas the 73-kDa band was still observed (Fig. 3B, left panel). Interestingly, arginine substitution at K194 led to the disappearance of both bands at 90 kDa and 73 kDa. These two bands were also not detected in the K119R/ K194R double mutant (2KR). Basically identical results were obtained in the presence of exogenously expressed GFP-SUMO-1. As shown in the right panel of Fig. 3B, the 250-kDa band was not observed with the
Komatsu et al. • Regulation of Ad4BP/SF-1 by SUMO
K119R mutant, and both the 250-kDa and 150-kDa bands disappeared in the K194R mutant and the 2KR double mutant. The failure of the sumoylated products to appear in the 2KR double mutant, together with the fact that these residues lie within sequences that conform to the sumoylation consensus, argues strongly that they constitute the actual sites of modification. In addition, interestingly, conjugation of SUMO at K194 was revealed to be essential for the second sumoylation at K119. In these assays, the band at 70 kDa observed in the wild type persisted, even when the mutant forms of Ad4BP/SF-1 were expressed, which indicated that the 70-kDa band represents unsumoylated Ad4BP/SF-1. Sumoylation of Ad4BP/SF-1 was further confirmed by an in vitro study. As shown in Fig. 4A, incubation of recombinant Ad4BP/SF-1 with SUMO-1, E1, and E2 produced the sumoylated forms of Ad4BP/SF-1. The two bands at 73 kDa and 90 kDa were thought to represent Ad4BP/SF-1 conjugated with one and two SUMO molecules, respectively. Sumoylation of Ad4BP/SF-1 did not occur when either E1, E2, or SUMO-1 was omitted. As expected, the 2KR mutant gave no sumoylated product (Fig. 4B). The function of PIAS was investigated with recombinant PIAS1 and PIAS3. As shown in Fig. 4C, addition of recombinant PIAS3 and, to a lesser extent, PIAS1, increased the efficiency of SUMO-1 conjugation to Ad4BP/SF-1. These effects of PIAS were similar to those observed in HEK 293 cells as described above. K194 Sumoylation Site Lies within the SC Region It has been shown that certain transcription factors synergistically activate transcription from a promoter containing multiple binding sites, and that the synergy is regulated through the SC motif (41). Recently, it was revealed that the SC motif consists of the sumoylation consensus sequence with proline residues at aminoand/or carboxy-terminal sides. Interestingly, because prolines are present at both sides, the sequence around K194 of Ad4BP/SF-1 satisfy SC motif consensus (41) (Fig. 5A). Thus, we used thymidine kinase (tk) reporter constructs containing one (Ad4x1) and three (Ad4x3) Ad4/SF-1 binding sites to determine whether the sequence found in Ad4BP/SF-1 functions as an SC motif. As expected, the wild-type Ad4BP/SF-1 synergistically activated transcription from the Ad4x3 reporter, leading to more transcription when compared with the Ad4x1 construct (Fig. 5B). Interestingly, transcription from the Ad4x3 reporter was further enhanced by approximately 3-fold with the 2KR mutant. This enhancement was also observed with the K194R mutant. In contrast, K119R, which is still capable of being sumoylated at K194, failed to show the mutantassociated enhanced synergy. Because it was indicated in the present study that PIAS1 and PIAS3 interact with and enhance sumoylation of Ad4BP/SF-1, the effects of PIAS1 and PIAS3 on the transcriptional activity of Ad4BP/SF-1 were exam-
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Fig. 4. In Vitro Sumoylation of Ad4BP/SF-1 A, In vitro sumoylation of wild-type Ad4BP/SF-1. Ad4BP/ SF-1 synthesized in a vaculovirus expression system was incubated in the presence or absence of E1, E2, and SUMO-1 for 3 h at 30 C as described in Materials and Methods. The reactions were terminated by the addition of sample buffer for SDS-PAGE and subjected to Western blot analysis with antiAd4BP/SF-1 antibody. B, In vitro sumoylation of 2KR-mutant Ad4BP/SF-1. The wild-type and 2KR-mutant Ad4BP/SF-1 were synthesized in a vaculovirus expression system. They were incubated with E1, E2, and SUMO-1 and subjected to Western blotting under the same conditions as described in panel A. C, Enhancement of sumoylation of Ad4BP/SF-1 by PIAS1 and PIAS3. Ad4BP/SF-1 was incubated in the presence or absence of recombinant Flag-PIAS1 or PIAS3 synthesized in a vaculovirus expression system and analyzed by Western blot. Arrow indicates the unmodified form of Ad4BP/ SF-1, whereas single and double closed arrowheads indicate forms modified with SUMO-1 at one and two lysine residues, respectively.
ined. PIAS3 repressed the transcriptional activity of Ad4BP/SF-1 in a dose-dependent manner, to a maximum suppression of 50% (Fig. 5C). Repression by PIAS3 was also observed with the K194R mutant. However, when 20 ng of the PIAS3 expression vector were transfected, the repression of transcription in the presence of the K194R mutant was statistically less than that of the wild type, whereas 50 and 100 ng PIAS3 repressed the activity of the K194R mutant as efficiently as they did the wild type. PIAS1 showed a similar pattern of repression as PIAS3 (data not
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Fig. 5. Synergistic Transcriptional Activity Enhanced by KR Mutation at Sumoylation Sites in Ad4BP/SF-1 A, Sumoylation consensus overlapped with SC motif. The consensus sequence for the SC motif consists of a sumoylation consensus with prolines on either or both sides. The K194 sumoylation site of Ad4BP/SF-1 agrees with the SC motif consensus. The amino acid residues corresponding to the SC motif consensus are underlined. ⌿ indicates a hydrophobic amino acid. B, Effects of KR mutation on synergistic transcription by Ad4BP/SF-1. Expression plasmids for the wild-type and KR mutants (K119R, K194R, and 2KR) of Ad4BP/SF-1 (20 ng) were transfected into HEK 293 cells with a luciferase reporter gene containing one (Ad4x1) or three Ad4/SF-1 sites (Ad4x3) upstream of the tk basal promoter (50 ng). Simultaneously, the expression vector for -galactosidase (5 ng) was transfected to normalize transfection efficiencies. Results are means ⫾ SD of triplicate transfections. Relative luciferase activity normalized by -galactosidase activity is expressed numerically. C, Effects of PIAS3 on the transcriptional activities of the wild-type and K194R mutant of Ad4BP/SF-1. Expression plasmids for the wild-type and K194R-mutant Ad4BP/SF-1 (20 ng) were transfected into 293 cells with increasing amounts of 3xFlag-PIAS3 (20, 50, 100 ng), a luciferase reporter gene containing three Ad4/SF-1 sites (Ad4x3-tk) (50 ng), and -galactosidase (5 ng). Results are means ⫾ SD of triplicate transfections. Relative luciferase activity normalized by -galactosidase activity is expressed as a percentage of that obtained in the absence of PIAS3 (9.3 ⫾ 0.6 for the wild type and 23.5 ⫾ 1.3 for the K194R mutant). *, P ⬍ 0.05.
shown). Likewise, both forms of PIAS repressed the transcription mediated by Ad4BP/SF-1 from other gene promoters, such as those that control the P450SCC (CYP11A1) and Mis genes (data not shown). Differential Implication of SC Motifs in Sox9, Gata4, and Wt1 in the Synergistic Control of Mis Gene Transcription It has been established that the transcription of the Mis gene is regulated by multiple transcription factors,
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including Sox9, Gata4, and Wt1 in addition to Ad4BP/ SF-1, and that Ad4BP/SF-1 activates Mis gene transcription synergistically with these factors through direct physical interaction (13–16). Thus, in the present study, we examined the possibility that this transcriptional synergy is controlled by the SC motif and/or sumoylation. Indeed, it was indicated by a previous study that Sox9 harbors SC motif (41), whereas the primary structures indicate that Gata4 and Wt1 also harbor SC motifs (Fig. 6A). Therefore, we examined whether the lysine residues within these sequences are potential sumoylation targets. Investigation with cultured cells showed that Sox9 and Gata4 could be sumoylated at a single site (Fig. 6B). The modified signal disappeared in cells transfected with the K396R mutant of Sox9. K366R Gata4 mutant-expressing cells also did not exhibit a modified signal, although much less protein was expressed than in cells harboring the wild-type plasmid. In the case of Wt1, three modified signals were observed in wild-type-expressing cells (indicated by closed arrowheads). All these signals
Fig. 6. Sox9, Gata4, and Wt1 as Sumoylation Targets A, Sequence alignment of the candidates for sumoylation and SC motifs found in Sox9, Gata4, and Wt1. The amino acid residues corresponding to the SC motif consensus are underlined. ⌿ indicates a hydrophobic amino acid. B, Identification of sumoylated products. Expression plasmids for Sox9, Gata4, Wt1, and Flag-tagged Dax1 were transiently transfected into HEK 293 cells in the presence or absence of GFP-SUMO-1. Total cell lysates were immunoblotted with anti-Sox9, anti-Gata4, anti-Wt1, and anti-Flag antibody, respectively. The sumoylated products are indicated by closed arrowheads. In the case of Wt1, a new signal appeared with the KR mutant as is indicated by an open arrowhead.
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disappeared in cells expressing K73R mutant Wt1, and another possibly sumoylated signal was detected (indicated by an open arrowhead). These results strongly suggest that Sox9, Gata4, and Wt1 are all sumoylated predominantly at the lysine residues in their SC motifs. Dax-1, which acts as a repressor of Ad4BP/SF-1, was not sumoylated. Considering these observations, we next examined whether these SC motifs regulate synergistic activation with Ad4BP/SF-1. The transcriptional synergy between Ad4BP/SF-1 and the other factors was investigated with the Mis reporter gene. As shown in Fig. 7B, neither wild-type nor K396Rmutant Sox9 showed clear activation in isolation, whereas Ad4BP/SF-1 activated transcription by 5-fold, with the activity of the K194R mutant slightly higher than that of the wild type. The wild types of Ad4BP/SF-1 and Sox9 synergistically activated transcription as reported previously (13). The synergy was enhanced when either the Ad4BP/SF-1 or the Sox9 KR mutant was used together with wildtype Sox9 or Ad4BP/SF-1, respectively. Moreover, as expected, this synergy was further enhanced when the KR mutants of both Ad4BP/SF-1 and Sox9
Fig. 7. Enhancement of Synergistic Activity by KR Mutants of Ad4BP/SF-1 and Sox9 A, Schematic presentation of promoter structure of the mouse Mis gene. cis-Acting elements 5⬘ upstream of the mouse Mis gene are indicated. B, Effects of KR mutation on the synergistic transcription between Ad4BP/SF-1 and Sox9. Expression plasmids for wild-type or K194R-mutant Ad4BP/ SF-1 (20 ng), wild-type or K396R-mutant Sox9 (20 ng), a luciferase reporter gene containing the Mis gene promoter (50 ng), and -galactosidase (5 ng) were transfected into HEK 293 cells. Luciferase activity was normalized by -galactosidase activity. The results are means ⫾ SD of triplicate transfections. C, Effects of KR mutation on the synergy between Ad4BP/SF-1 and Wt1. Expression plasmids for wild-type or K194R-mutant Ad4BP/SF-1 (10 ng) and wild-type or K73Rmutant Wt1 (100 ng) were transfected into HEK 293 cells together with the luciferase reporter gene containing the Mis gene promoter (50 ng) and -galactosidase (5 ng).
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were transfected simultaneously. On the other hand, although Ad4BP/SF-1 acts with Wt1 to synergistically activate Mis gene transcription, this synergy was not affected by the presence of any of the KR mutants (Fig. 7C). Because the amount of the Gata4 KR mutant expressed in the cultured cells was significantly less than that of the wild type (Fig. 6B), the effect of the KR mutation on transcriptional synergy between Ad4BP/SF-1 and Gata4 was not investigated. DNA Binding and Protein Interaction of Ad4BP/ SF-1 Is Unaffected by Sumoylation Considering that the lysine residues in the Ad4BP/ SF-1 and Sox9 SC motifs have potential as sumoylation targets, the studies above strongly suggested that sumoylation suppresses the synergistic transcription between Ad4BP/SF-1 and Sox9. Therefore, it was assumed that the DNA binding activity of sumoylated Ad4BP/SF-1 is weaker than that of the unsumoylated form and/or the physical interaction activity of sumoylated Ad4BP/SF-1 with Sox9 is weaker than that of the unsumoylated form. To test these possibilities, an in vitro sumoylated form of Ad4BP/SF-1 was used for the following studies. When DNA binding activity was examined by EMSA, a retarded signal was detectable even when the sumoylated form of Ad4BP/SF-1 was used, although their signal was broad compared with that of the unsumoylated form (Fig. 8A). An antibody against SUMO-1 gave a supershifted signal when applied to the sumoylated material, whereas an antibody to Flag gave supershifted signals with both the sumoylated and unsumoylated forms of Ad4BP/SF-1. The effect of sumoylation on DNA binding was also examined with a DNA pull-down assay. As shown in Fig. 8B, sumoylated Ad4BP/SF-1 was recovered as efficiently as was the unsumoylated form. The effect of sumoylation on physical interaction was examined by a pulldown assay using recombinant His-Sox9 (Fig. 8C). An equal amount of unsumoylated and sumoylated Ad4BP/SF-1 was recovered through their interaction with Sox9. These results indicate that the effects of SUMO-1 modification of Ad4BP/SF-1 do not result from disturbance of DNA binding and interaction with Sox9 and thus suggested that the effects of SUMO-1 modification is mediated by undefined factors. In this regard, it was interesting to note that DEAD box-containing DP103 interacts with Ad4BP/SF-1 around K194 and thereby represses transcription (23). Thus, we tested the possibility that interaction with DP103 is affected by the KR mutation at 194 using a coimmunoprecipitation assay. When the HEK 293 total-cell lysates transfected with 3xFlag-DP103 and GFP-Ad4BP/SF-1 were subjected to immunoprecipitation with an anti-Flag antibody, both wild-type and K194R-mutant Ad4BP/SF-1 were recovered with similar efficiency (Fig. 9A). Moreover, the repression of transcriptional activity of K194R by DP103 was comparable to that of the wild type (Fig. 9B). Therefore, the
Fig. 8. Effects of Sumoylation on DNA Binding and Protein Interaction Activity of Ad4BP/SF-1 A, DNA binding activity of sumoylated and unsumoylated Ad4BP/SF-1 revealed by EMSA. Recombinant Flag-Ad4BP/ SF-1 was subjected to a sumoylation reaction in the presence or absence of SUMO-1. The extent of sumoylation is indicated as an input in panel B. The DNA binding activities of sumoylated and unsumoylated Ad4BP/SF-1 were determined by EMSA with an oligonucleotide containing an Ad4/ SF-1 site. Arrowheads indicate protein-DNA complexes. To confirm the specificity of the signal, anti-SUMO-1 and antiFlag antibodies were added to the binding mixtures. The supershifted complexes with anti-SUMO-1 and anti-Flag antibodies are indicated by single and double asterisks, respectively. B, DNA binding activity of sumoylated and unsumoylated Ad4BP/SF-1 revealed by DNA pull-down assay. Sumoylated or unsumoylated Flag-Ad4BP/SF-1 was incubated with a biotinylated oligonucleotide containing an Ad4/ SF-1 site. Proteins bound to the oligonucleotide were precipitated with avidin-coated beads, followed by Western blotting with anti-Flag antibody. An arrow indicates unsumoylated Ad4BP/SF-1, and single and double arrowheads indicate forms sumoylated at one and two lysine residues, respectively. C, Effect of sumoylation on interaction between Ad4BP/SF-1 and Sox9. After sumoylated or unsumoylated Flag-Ad4BP/SF-1 was incubated with recombinant HisSox9, Sox9 was recovered with nickel beads. Coprecipitated Ad4BP/SF-1 with Sox9 was detected with anti-Flag antibody.
enhancement of transcriptional activation by the K194R mutant is independent of a DP103-related repression.
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Fig. 9. Effect of K194R Mutant of Ad4BP/SF-1 on Interaction and Suppression by DP103 A, Interaction of DP103 with wild-type and K194R-mutant Ad4BP/SF-1. Expression plasmids for wild-type or K194Rmutant GFP-Ad4BP/SF-1 and 3xFlag-DP103 were transfected into HEK 293 cells. Total cell lysates were subjected to immunoprecipitation with anti-Flag antibody. Coimmunoprecipitated GFP-Ad4BP/SF-1 was detected with anti-Ad4BP/ SF-1 antibody. Ad4BP/SF-1 in 2% of total cell lysates are indicated. B, Suppression of transcription mediated by wildtype and K194R-mutant Ad4BP/SF-1 by DP103. Expression plasmids for wild-type and K194R-mutant Ad4BP/SF-1 (20 ng) were transfected into HEK 293 cells with increasing amounts of HA-DP103 (100, 200, 500 ng), a luciferase reporter gene containing three Ad4/SF-1 binding sites (50 ng), and a -galactosidase expression vector (5 ng). Results are means ⫾ SD of triplicate transfections. Luciferase activity normalized by -galactosidase activity is expressed as a percentage of that obtained in the absence of DP103 (8.9 ⫾ 0.6 for wild type and 24.5 ⫾ 0.5 for K194R mutant).
DISCUSSION Functional Regulation of Ad4BP/SF-1 through the Hinge Region Ad4BP/SF-1 is unique within a nuclear receptor superfamily in possessing a long hinge region between its Zn finger DBD and putative ligand binding domain (LBD). The hinge region consists of approximately 140 amino acids and has a proline-rich segment (45). Functionally, the amino-terminal 30 amino acids in the hinge region, designated as the Ftz-F1 box, was reported to cooperate with the DBD in nucleotide recognition (46). In addition, a bipartite nuclear localization signal also resides within the hinge region (47). DP103, a DEAD box-containing molecule, acts as a suppressor of Ad4BP/SF-1-mediated transcription by
Komatsu et al. • Regulation of Ad4BP/SF-1 by SUMO
interacting with a repression domain located at amino acids 193–201 within the hinge region (23). In addition, it was described that recruitment of transcriptional cofactors was regulated by phosphorylation at Ser 203 (27). Taken together, these lines of evidences have clearly defined the hinge region as crucial for the diverse functions of Ad4BP/SF-1. In the present study, in vivo and in vitro experiments demonstrated that Ad4BP/SF-1 is a target for sumoylation at two acceptor sites, K119 and K194, both of which are located within the hinge region. Interestingly, sumoylation at K194 is strictly required for subsequent sumoylation of K119. Such an ordered process of sumoylation was also described with c-Myb (48). With these transcription factors, the ordered sumoylation strongly suggests that the first results in a conformational change that enables the sumoylation of the next site. In the case of Ad4BP/SF-1, phosphorylation at Ser 203 in the hinge region was shown to render the LBD structure more stable and compact, similar to ligand-dependent receptor activation (28). Thus, it is assumed that modification with SUMO within the hinge region potentially regulates the transcriptional activity of Ad4BP/SF-1 through conformational changes. Generalized Function of the SC Motif It has been frequently observed that certain types of transcription factors activate transcription synergistically when their binding sites are present in multiple copies in a promoter region. Regarding the molecular mechanisms underlying synergistic transcriptional activation, Iniguez-Lluhi and Pearce (41) identified a unique amino acid sequence and designated it an SC motif. This SC motif is present within negative regulatory and attenuator domains of several transcription factors, including that of Ad4BP/SF-1. However, the function of the putative SC motif around K194 remains to be investigated. In the present study, we have used a synthetic promoter containing multiple Ad4/SF-1 sites and found that the sequence in Ad4BP/SF-1 acts as the SC motif. The SC motif has been identified so far using synthetic and natural promoters harboring multiple binding sites of a single transcription factor such as the glucocorticoid receptor (41) and CCAAT enhancer binding protein-␣ (42). However, synergistic transcription has been seen frequently to occur between distinct types of transcription factors on natural gene promoters. Therefore, we examined whether the SC motif of Ad4BP/SF-1 affects its transcriptional synergy with other types of transcription factors. For this purpose, the Mis gene was used as a luciferase reporter construct because its gene transcription is controlled synergistically by Ad4BP/SF-1 through interaction with Sox9, Gata4, and Wt1 (13–16). Because the SC motif was found to be crucial for synergistic activation of Ad4BP/SF-1, we searched for SC motifs in Sox9, Gata4, and Wt1 and found one in each of the three
Komatsu et al. • Regulation of Ad4BP/SF-1 by SUMO
proteins. Thus, we examined whether these SC motifs are involved in the synergistic transcription of the Mis gene and found that the synergy between Ad4BP/ SF-1 and Sox9 is, in fact, regulated by the SC motifs in both factors. Although the function of the SC motif has been discussed in mediating transcriptional synergy between identical factors, it has also been suggested that SC motif is implicated in transcriptional synergy between two distinct factors (42, 43). Actually, the SC motif in Drosophila activator Dorsal was reported to regulate the transcriptional synergy with another factor Twist (49). In the present study, we found that the SC motifs in two distinct factors, Ad4BP/SF-1 and Sox9, are both involved in the regulation of transcriptional synergy on a natural promoter context. Taken together with the fact that the SC motif is frequently found in transcription factors, these findings imply that the SC motif potentially provides a molecular basis for generalized mechanisms of transcriptional regulation, even though it was originally characterized in a limited context. Surprisingly, however, the SC motif of Ad4BP/SF-1 did not seem to be involved in its transcriptional synergy with Wt1. In general, it is well accepted that synergistic transcription is mediated by proteinprotein interaction, and the interaction of Ad4BP/SF-1 with Wt1 was demonstrated previously (15). For ciselements, it was reported that synergistic activity is largely influenced by the distance between the two binding sites (50), suggesting that the promoter structure is important for the synergy to occur. Accordingly, implication of the SC motif in the synergy of Ad4BP/ SF-1 with Wt1 may be possible in their regulation of other genes with more appropriately spaced binding sites. Alternatively, the cooperation of SC motif belonging to two different types of transcription factors may be selective and preferential depending on the particular combination of transcription factors, even though they both generally show synergistic transcription. It remains to be seen, however, whether Wt1 shows SC motif-regulated synergy with other factors. Possible Implication of Sumoylation in Synergistic Transcriptional Regulation When discussing the function of the SC motif, it should be noted that the motif overlaps with the sumoylation consensus sequence (41, 42). To study this feature, Holmstrom et al. (43) investigated the correlation between SC motif function and sumoylation and showed that sumoylation of the SC motif leads to transcriptional suppression. In agreement with this finding, transcriptional activity was found to be decreased when transcription factors were artificially fused to SUMO (36, 43, 51). In the case of Ad4BP/SF-1, the present study showed that K194 in the SC motif can, in fact, be sumoylated, and mutation of this lysine residue leads to simultaneous loss of SC motif function and sumoylation, strongly suggesting that the SUMO molecule is required for SC motif function.
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It has been proposed as a mechanism of regulation by the SC motif that a putative synergy control factor (SCF) is recruited by the SC motif, and this binding of SCF to the SC motif is regulated by sumoylation (41, 42). To detect the possible presence of an SCF, we examined whether SUMO affects directly the DNA binding and Sox9 interaction of Ad4BP/SF-1 using in vitro sumoylated Ad4BP/SF-1. The lack of effect on either activity suggests that transcriptional suppression by SUMO occurs via the function of another unknown protein, such as a putative SCF, and not via a direct effect on the function of the target protein. This finding suggested the possibility that a DEAD boxcontaining protein, DP103, which was identified as a factor interacting with the repressor domain carrying the SC motif of Ad4BP/SF-1(23), acts as such an SCF. However, the interaction between Ad4BP/SF-1 and DP103 was not altered by the K194R mutation and, moreover, suppression of wild-type and K194Rmutant Ad4BP/SF-1-mediated transcription by DP103 was identical. Thus, it is unlikely that DP103 acts as the SCF. As the candidate for the SCF, it is interesting to note that histone deacetylase (HDAC) is recruited to the sumoylated forms of Elk-1 (52) and p300 (34), leading to suppression of transcription mediated by these factors. The role of HDAC and identification of other possible factors involved in sumoylationmediated transcriptional suppression of Ad4BP/SF-1 should be addressed in the future. Bipartite Functions of PIAS It has been discussed that PIAS suppresses transcriptional activity through both sumoylation-dependent and sumoylation-independent pathways (53, 54). Because both PIAS1 and PIAS3 function as the E3 ligases for sumoylation of Ad4BP/SF-1, PIAS may be implicated in transcriptional regulation of Ad4BP/SF-1 through a sumoylation-dependent pathway. However, PIAS3 still suppressed the activity of the KR mutant, indicating the presence of a sumoylation-independent pathway for Ad4BP/SF-1 activity. To elucidate the mechanism for the sumoylation-independent pathway, we investigated whether PIAS affects subnuclear localization of wild-type and KR-mutant Ad4BP/SF-1. Distributions of both types of Ad4BP/SF-1 were not affected by the presence of PIAS and/or SUMO (data not shown). Subsequently, considering that the DBD of Ad4BP/SF-1 is involved in interaction with PIAS, we used vacculovirus-synthesized proteins to investigate the effect of PIAS on the DNA binding activity of Ad4BP/SF-1. However, PIAS again had no effect on the DNA binding activity (data not shown). Thus, it is possible to assume that PIAS affects transcriptional activity through interaction with corepressors such as HDAC (55, 56). In this study, we showed that Ad4BP/SF-1 is sumoylated at two lysine residues and found evidence that sumoylation at the SC motif regulates the transcriptional activity of Ad4BP/SF-1. However, the fine
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molecular mechanisms underlying regulation by SUMO remain unclear. The identification of novel factors that interact preferentially with SUMO would provide valuable insight into the myriad functions of SUMO.
MATERIALS AND METHODS Plasmids Mammalian expression plasmids for mouse Ad4BP/SF-1 were constructed by subcloning the cDNA provided by Dr. Keith Parker (University of Texas Southwestern Medical Center, Dallas, TX) into pCMX (57), pEGFP-C1 (CLONTECH Laboratories, Palo Alto, CA), and p3xFlag-CMV10 (Sigma Chemical Co., St. Louis, MO). Sox9, Gata4, and Wt1 isolated from a mouse fetal gonad cDNA library were subcloned into pRC/ RSV (Invitrogen, Carlsbad, CA). Wt1 used in this study was a splice variant lacking the KTS amino acids (15, 58). K119 and K194 in Ad4BP/SF-1, K396 in Sox9, K366 in Gata4, and K73 in Wt1 were substituted with arginines by PCR. The expression plasmids, pEGFP-SUMO-1 and pEGFP-SUMO-2, were kindly provided by Dr. Hisato Saitoh (Kumamoto University, Kumamoto, Japan). Expression plasmids for 3xFlag-tagged PIAS1 and PIAS3 were constructed using cDNAs obtained by yeast two-hybrid screening. DP103, generously provided by Dr. Yoel Sadovsky (Washington University School of Medicine, St. Louis, MO) was subcloned into p3xFlag-CMV10 and pHA-CMX. A luciferase reporter plasmid containing the enhancer region of the Mis gene was constructed by inserting the 380 bp upstream from the transcription initiation site into pGL3-Basic (Promega Corp., Madison, WI). The luciferase reporter plasmids, Ad4x1 and Ad4x3, have one and three Ad4/SF-1 sites, respectively, located 5⬘ upstream of the tk basal promoter (25). Cell Culture, Transient Transfection, and Reporter Assay HEK 293 cells were grown in DMEM (Sigma) supplemented with 10% fetal bovine serum (JRH Sciences, Lenexa, KS) and 1⫻ penicillin-streptomycin-glutamine (Invitrogen) at 5% CO2 and 37 C. According to the manufacturer’s protocol, 6 ⫻ 104 cells were seeded in 24-well plates 24 h before transfection with lipofectamine reagent (Invitrogen). The total amount of the transfected plasmids was adjusted to 375 ng with the vector plasmids. pCMV-SPORT--gal (Invitrogen) was used as an internal control to normalize transfection efficiency. The cells were harvested 36 h after transfection, and the cell lysates were subjected to luciferase and -galactosidase assays as described previously (25). All transfection experiments were performed in triplicate. Immunoprecipitation and Western Blotting To detect the sumoylated forms of Ad4BP/SF-1, Sox9, Gata4, and Wt1 in cultured cells, the expression plasmids were transfected into HEK 293 cells for 48 h with the expression plasmid for pEGFP-SUMO-1 or SUMO-2. The cells were lysed directly with SDS-PAGE sample buffer and thereafter were subjected to Western blot analysis with anti-Ad4BP/ SF-1 antibody (59), anti-Sox9 antibody provided by Dr. Peter Koopman (The University of Queensland, Brisbane, Australia), anti-Gata4 antibody (C-20, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-Wt1 antibody (C-19, Santa Cruz Biotechnology), and anti-SUMO-2/3 antibody (Dr. Hisato Saitoh). For immunoprecipitation, the cells transfected with the expression vectors were lysed in a lysis buffer containing
Komatsu et al. • Regulation of Ad4BP/SF-1 by SUMO
50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 1% Triton X-100, 10% glycerol, 10 mM N-ethylmaleimide, complete protease inhibitor cocktail (Roche, Mannheim, Germany), 1 mM NaF, 2 mM sodium orthovanadate, 25 mM -glycerophosphate, and 10 mM sodium pyrophosphate. After centrifugation at 15,000 rpm at 4 C for 10 min, the supernatants were diluted 2-fold with lysis buffer without NaCl and incubated with anti-GFP antibody (Medical & Biological Laboratories, Nagoya, Japan) or anti-Flag M2 antibody-agarose (Sigma) at 4 C for 1 h. Immune complexes with anti-GFP antibodies were recovered with protein A sepharose (Amersham Biosciences, Uppsala, Sweden). The immunoprecipitates were analyzed by SDS-PAGE, followed by Western blotting with anti-Flag M2 antibody or anti-Ad4BP/ SF-1 antibody. In Vitro Sumoylation, Pull Down Assay, and EMSA Flag-Ad4BP/SF-1, Flag-PIAS1, Flag-PIAS3, and His-Sox9 were expressed by recombinant baculoviruses in Sf21 cells according to the manufacturer’s protocol (Invitrogen). The cells were lysed in 20 mM Tris-HCl (pH 8.0), 400 mM KCl, 10% glycerol, 5 mM MgCl2, 0.1% Tween 20, and complete protease inhibitor cocktail (Roche). The Flag-tagged proteins were recovered with anti-Flag M2 antibody-agarose. The agarose was washed with the buffer above containing only 100 mM KCl, and the recovered proteins were eluted with 150 g/ml 3xFlag peptide. His-Sox9 was recovered with nickel beads (Invitrogen) and eluted with 300 mM imidazole. Aos1 fused with Uba2 in tandem was synthesized as a fusion protein with glutathione-S-transferase (GST) (mAU/ pGEX-KG) in BL21(DE3)Star. After the Escherichia coli was cultured at 30 C, 0.02 mM isopropyl--D-thiogalactopyranoside (IPTG) was added to induce expression. The E. coli was sonicated in PBS containing 1 mM dithiothreitol (DTT), and the fusion protein (E1) was recovered by glutathione sepharose 4 FF (Amersham Biosciences). The purified E1 was dialyzed against 25 mM HEPES-KOH (pH 7.5), 50 mM KCl, 1 mM DTT, and 5% glycerol. Xenopus Ubc9 was cloned into pET28a for expression as a His-tagged protein, His-Ubc9 (His-Ubc9/pET28). His-Ubc9 was synthesized in BL21(DE3) cells at 37 C with IPTG (0.4 mM) induction. The E. coli was recovered and then sonicated in 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, and 2 mM 2-mercaptoethanol. Then, the sonicate was loaded onto a diethylaminoethyl-sepharose FF (Amersham Biosciences) column, and the flow-through fraction was used for purification with nickel-chelating sepharose FF (Amersham Biosciences). The eluate with imidazole was dialyzed against the buffer used for dialysis of the E1 protein. SUMO-1 (1–97 amino acid) was inserted into pGEX-4TK for expression as a GST-fusion protein in BL21(DE3). The recombinant E. coli was cultured at 37 C followed by IPTG (0.4 mM) induction. The E. coli was sonicated in PBS containing 1 mM DTT, and the sonicate was charged onto glutathione sepharose 4 FF. SUMO-1 was eluted by thrombin digestion of GST-SUMO-1 on the column. The recovered SUMO-1 was dialyzed against the buffer used for dialysis of the E1 protein. In vitro sumoylation was performed with the recombinant Flag-Ad4BP/SF-1 (0.5 M), E1 (0.2 M), E2 (His-Ubc9, 3 M), SUMO-1 (10 M), and PIAS1 or PIAS3 (0.5 M) in 60 l of a sumoylation buffer containing 25 mM HEPES (pH 7.5), 150 mM KCl, 4 mM MgCl2, 0.01% Triton X-100, 1 mM DTT, and 5 mM ATP. After the in vitro sumoylation mixture was incubated at 30 C for 3 h, the mixture was precleared with nickel beads to remove His-Ubc9. Subsequently, the mixture was incubated with recombinant His-Sox9 for 1 h on ice in a binding buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10% glycerol, 5 mM MgCl2, 0.1% Tween 20, and 20 mM imidazole. His-Sox9 was recovered using nickel beads, and the fraction was subjected to Western blot analysis for Ad4BP/SF-1. DNA pull down was conducted essentially as described elsewhere (25). EMSA was performed as described previously (1), using an oligonucleotide containing the Ad4/SF-1 consensus site
Komatsu et al. • Regulation of Ad4BP/SF-1 by SUMO
as the probe. For supershift assay, anti-SUMO-1 antibody (21C7; Zymed Laboratories, Inc., south San Francisco, CA) or anti-Flag M2 antibody was added to the binding mixture before the addition of the DNA probe.
Acknowledgments We thank Dr. Keith Parker for mouse SF-1 plasmid; Dr. Yoel Sadovsky for mouse DP103 plasmid; Dr. Peter Koopman for anti-Sox9 antibody; Dr. Hisato Saitoh for GFPSUMO1/2 plasmids and anti-SUMO-2/3 antibody; and Yuko Shinohara for technical assistance.
Received April 27, 2004. Accepted June 3, 2004. Address all correspondence and requests for reprints to: Ken-ichirou Morohashi, Prof., Ph.D., Department of Developmental Biology, National Institute for Basic Biology, Myodaijicho, Okazaki 444-8787, Japan. E-mail:
[email protected]. This work was supported in part by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology. Note Added in Proof SUMO-1 modification of Ad4BP/SF-1 has also been observed by Chung and colleagues (60).
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